LncXIST Facilitates Iron Overload and Iron Overload-Induced Islet Beta Cell Injury in Type 2 Diabetes through miR-130a-3p/ ALK2 Axis

Department of Geriatrics, e First People’s Hospital of Yunnan Province, e Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, China Medical Laboratory, e First People’s Hospital of Yunnan Province, e Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, China Department of Endocrine, e First People’s Hospital of Yunnan Province, e Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, China


Introduction
Type 2 diabetes (T2D) is a metabolic disease mediated by environmental and genetic factors, accounting for more than 90% of the total prevalence of diabetes, and is associated with increased risk of colon, uterine, liver, pancreatic, breast (in postmenopausal women), and prostate cancers amongst others [1]. At present, it is generally believed that dysfunction and insulin resistance (IR) of β-cell of the pancreatic islet are two important factors in the pathogenesis of T2D pathophysiology [2]. Since 1865, it was found that the risk of T2D increases in patients with hemochromatosis [3]. e association between iron overload and T2D has been paid more attention. Histopathological and clinical findings in earlier research provide evidence that iron overload impacts the pancreatic β-cell and affects insulin secretion, finally leading to diabetes [4]. Subsequently, a considerable number of studies have found that the majority of patients with T2D or T1D or gestational diabetes mellitus have dysmetabolic iron [5,6]. Hepcidin, a 25-amino acid peptide produced by the hepatocytes, is a key regulator of iron metabolism and responsible for iron deposition [7], which is dysregulated in diabetic patients. Iron overload exhibits loss of β-cell mass and increases hepatic glucose production and metabolic inflexibility [8,9]. e subsequent loss of islet β-cell can then lead to a gradual loss of functional capacity and decreased insulin sensitivity [10,11]. Recently, research demonstrated the association of iron homeostasis with IR in high-fat feeding mice [12]. However, the mechanism underlying the facilitation of iron on the development of T2D is still unknown.
Long noncoding RNA (lncRNA) is longer than 200 nucleotides, lacking defined protein coding potential. LncRNAs have emerged as key components of regulatory genome organization and gene expression. It is increasingly recognized that lncRNAs play an important function in the epigenetic processes in both health and disease [13,14]. In recent years, lncRNAs are considered to be regulatory molecules in islet function and the etiology of diabetes, such as lncHI-LNC25 [15], lncH19 [16], and lncMALAT1 [17]. Moreover, altered lncRNAs signatures are found in T2D patients, including increasing expression levels of XIST [18] in PBMCs from T2D patients compared to control subjects. Besides, XIST is linked with IR and inflammation in T2D [18], also participating in diabetic cardiomyopathy [19] and diabetic nephropathy [20]. In addition, lncRNAs also mediate intracellular iron metabolism. PVT1 influenced the uptake level of cellular iron in hepatocellular carcinoma, and its upregulation induced an iron metabolism disorder and promoted tumorigenesis [21]. e relationship between XIST and iron overload in T2D is not well known.
In this paper, we have proposed a novel approach and verified its applicability through various experiments. e results showed that XIST overexpression in iron overload rats was associated with a coincident alteration in iron-related parameters. In vitro, increasing XIST level in response to iron-induced was associated with iron metabolism, proliferation inhibition, apoptosis, and reduction of insulin secretion. In contrast, XIST knockdown alleviated above islet β-cell damage induced by iron overload. e remaining paper is organized as follows. In the subsequent section, various groups of animals, which are formed to verify the operational superiority of the proposed scheme, are described along with various metrics. Moreover, treatment methods were described along with feasible duration. In Section 3, performance evaluation of the proposed scheme in terms of various metrics has been presented. Finally, summary of the proposed approach is presented in the conclusion section.

Animals Group Treatment.
A total of 25 male Wistar rats weighing between 240 and 250 g (aged 7 weeks) were from the Experimental Animal Center of Southern Medical University (Guangzhou, China). All rats were housed in static cages with controlled ambient temperature (22-25°C) on a 12-h light/dark cycle. After 1 week of acclimatization, the animals were randomly assigned to four groups: the control group (C) and iron overload group (IO) were fed with normal pellet diet (NPD) and five doses of intraperitoneal injection of 60 mg/kg iron-dextran-saline (ferric hydroxide dextran complex; Sigma, St. Louis, MO, USA) as previously described [22]. e T2D (D) and iron overload-related T2D (DIO) group were fed with the highfat diet and injected with 35 mg/kg streptozotocin (STZ; Sigma, St. Louis, MO, USA) in the 4th week. For DIO group, the iron-dextran-saline was administrated with iron dextran saline, the same as IO group. e rats in all groups were weighed each week, and they were euthanized under ether anesthesia at 10 weeks after STZ injection. Before killing the rats, the blood glucose levels were measured by glucose test strips (Roche Diagnostic, Indianapolis, IN, USA). All protocols of animals and experiments were checked and appropriated by Animal Research Ethical Committee of Kunming University of Science and Technology (Kunming, China).

Determination of Glycolic Levels.
On the 6th week and the last day of the treatment, the blood glycolic levels were measured to determine the success of diabetes induction using fresh blood with glucose test strips (Roche Diagnostics) according to the manufacturer's protocol.

Iron Parameters in Serum.
Serum iron concentrations and total iron-binding capacity (TIBC) in nonhemolyzed serum samples were made by colorimetric analysis kits (Nanjing Jian Cheng Biotechnology Institute, Nanjing, China). In addition, serum ferritin was determined in nonhemolyzed serum samples using ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China).

Terminal Dexynucleotidyl Transferase-Mediated dUTP
Nick End Labeling (TUNEL) Assay. According to the manufacturer's protocol, we have used TUNEL assay kit (Beyotime Company, Shanghai, China) for evaluating levels of cell apoptosis. Initially, every cell was fixed in 4% paraformaldehyde and permeabilized using 0.1% Triton X-100, and this process is repeated for every cell. After that, the cells were incubated in TUNEL reaction mixture for 1 h at 37°C. Nuclei were counterstained with DAPI, and the cells were observed with a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).

Cell Culture and DIO Treatment.
e rat INS-1 cells were purchased from Beijing Beina Chuanglian Biotechnology Institute (Beijing, China). Cells were cultured in RPMI1640 ( ermo Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; ermo Fisher Scientific, Inc.), 50 µM β-mercaptoethanol, and 1% penicillin and streptomycin (Gibco). Cells were incubated in a humidified atmosphere containing 5% CO 2 at 37°C [23]. e medium was renewed once per two days, and cells were passaged once per 6 to 7 days. INS-1 cells in the logarithmic growth phase were used for the following tests. e control cells (NC) were cultured with 25 mmol/L glucose, which were treated with 12.5, 25, 50, 100, or 200 μM iron-D for 48 h, respectively. At the end of the experiments, cell viability was determined.

2
Computational Intelligence and Neuroscience 2.6. Cell Transfection. Transfections were performed using miR-130a-3p mimic, miR-130a-3p inhibitor, and miRNA negative control, which were obtained from Shanghai GenePharma (Shanghai, China). e XIST and activin receptor-like kinase 2 (ALK2) overexpression vector (pc-XIST and pc-ALK2, respectively) were constructed to promote the expression of XIST and ALK2 in INS-1 cells, and the empty vector was used as a control. e XISTsmall interfering RNA was used to knock XIST expression. Cell transfection with the miRNA mimic, miRNA inhibitor, vectors, and siRNA was performed by using Lipofectamine 3000 (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol.

Real-Time Quantitative PCR Analysis (RT-qPCR).
In these experiments, total RNA was extracted with TRIzol solution (Takara Biotechnology Co., Ltd., Dalian, Liaoning Province, China) and subsequently converted to cDNA with Reverse Transcription Kit (Takara) according to manufacturer's instruction. e levels of XIST, miR-130a-3p, GAPDH, and U6 were determined using SYBR Premix Ex TaqTM II kit (TaKaRa) and SYBR Premix Ex Taq miRNA kit (TaKaRa). e relative expression of XIST and miR-130a-3p was calculated using 2 −ΔΔCt method [24], whereas GAPDH and U6 were used as the reference gene.

Cell Viability Analysis.
e viability of INS-1 cells was detected by using MTT kits (Sangon Biotech). Briefly, INS-1 cells were with RPMI-1640 and seeded into 96-well plates (2000 cells/well). e plate was incubated for 24 hours in a humidified incubator. 10 μL MTT reagent was added at particular time points for 4 h at 37°C. e optical density (OD) was measured at 570 nm with a micrometer reader (BioTek Instruments Inc.). e supernatants were centrifuged at 3000 rpm for 3 min, and then the insulin concentration was measured with ELISA kit (Abcam).

Labile Iron Pool (LIP) Evaluation.
As previously described, cellular content of LIP-Fe was measured by fluorospectrophotometry using calcein-AM ester [25]. Briefly, INS-1 cells were seeded in 96-well plates and washed twice with phosphate-buffered saline (PBS) and incubated with calcein-AM (0.25 mmol/L) for 30 min at 37°C, whereas the fluorescence intensity was measured by fluorospectrophotometer (Ex � 495 nm, Em � 530 nm) using IPP 6.0 software package. Following the incubation in the medium-containing desferrioxamine (DFO, 100 mmol/L) for 30 min at 37°C, the fluorescence intensity was measured again. e LIP was calculated as the difference in calcein-AM cells treated with or without DFO. e increased LIP level of hepatocytes following various treatments was expressed as fold change over control.

Haematoxylin and Eosin (H&E) Staining.
Pancreata tissues from each group rats were fixed in 10% neutral buffered formalin and processed for routine histology and paraffin embedding. Five micrometer tissue sections were stained with H&E and evaluated histologically to determine the degree of islet destruction. Staining of Brn3a was observed by Nikon Eclipse 80i microscope (Nikon Corporation).

Immunohistochemistry.
e tissue sections were baked and deparaffinized. Antigen retrieval was performed by boiling in sodium citrate buffer. After blocking endogenous peroxidase and nonspecific binding, tissue sections were incubated with primary (anti-insulin, no. ab181547, Abcam) and then with secondary (no. ab150077, Abcam) antibody. e sections were washed (3 times), and the reaction product was developed with diamino benzoate, counterstained with hematoxylene. Staining of Brn3a was observed by Nikon Eclipse 80i microscope (Nikon Corporation).

Luciferase Reporter Assays.
StarBase online software is used to predict the targeted binding sites of miRNAs in mRNAs and lncRNA. Here, we have predicted that the targeted binding relationships between miR-130a-3p and XIST or ALK2. We constructed reporter vectors, including wild-type (WT) and mutant-type (MUT) binding sites of XIST or ALK2 sequences, named as WT-XIST, MUT-XIST, WT-ALK2, and MUT-ALK2 and cotransfected with miR-130a-3p mimic or negative control into HEK 923 cells (ATCC, Manassas, VA, USA). Relative luciferase activity was assayed 48 h after transfection using the Dual-Luciferase Computational Intelligence and Neuroscience  Computational Intelligence and Neuroscience reporter assay system (Promega Corporation, Wisconsin, USA).

Statistical
Analysis. e data were expressed as mean ± standard error mean (SEM) and analyzed using Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Data between the two groups were analyzed by Student's ttest, and data among multiple groups were analyzed by oneway analysis of variance (ANOVA) with Dunnet's least significant difference post-hoc tests. P < 0.05 was regarded as statistically significant. e results were considered significant when P < 0.05.

Effect of Iron Overload on Body Weight, Fasting Blood
Glucose, Iron Parameters, and Pancreatic Islets/Beta Cells in T2D Rats. Compared with rats in the control group (NC), the individuals in T2D group (D) and iron overload-related T2D group (DIO) profiles had decreased body weights, while iron dextran injection only (IO) group showed no difference (Figure 1(a)). After 10th week, the weight gain of DIO group is slower than D group. Moreover, in D and DIO groups, rats performed a higher fasting blood glucose, and a more growth postprandial was observed (Figure 1(b)). To evaluate serum iron status, the serum iron and ferritin constituent and TIBC were measured. As shown in Figures 1(c)-1(e), three indexes were increased in IO and DIO groups. Next, the iron metabolic-related proteins were measured by Western blot, and results indicated that iron dextran-induced increased hepcidin, ferritin, and DMT1 proteins expression (Figure 1(f )). By contrast, the expression of TfR1 was decreased. Pancreatic morphology was analyzed to assess pathological changes. H&E staining revealed the well-defined islets of Langerhans, along with intact exocrine pancreatic tissues in control and IO groups, which were slightly lost in the sections from D group but seriously lost in the sections from DIO group (Figure 1(g), H&E staining). Sections of control and IO groups revealed dense reddish brown cytoplasmic immunoreactivity that was detected in the cells of the pancreatic islets of Langerhans. e immunoreactivity was observed in most of the islet cells ( Figure 1(g), insulin staining). Sections of D group revealed slightly increased immunoreactivity, but sections of DIO group revealed markedly decreased immunoreactivity that was detected in the central part of the islet of Langerhans. Finally, we detected the expression of XIST in different groups, and results showed that the level of XIST was significantly increased in pancreas tissues as well as the pancreas of DIO compared with D group (Figure 1(h)). Notably, iron overload-induced promoted XIST expression in T2D. e 50 μM iron-D markedly reduced the survival of the INS-1 cells. Hence, cells treated with 50 μM iron-D were used in the following study. To further explore the functions of XIST in INS-1 cells, we knocked the XIST in DIO cells (Figure 2(b)). en, the result of MTT assay demonstrated that knockdown of XIST significantly promoted cell proliferation (Figure 2(c)). e TUNEL assay showed that the apoptotic cell in XIST knockdown group was less than that in DIO + sh-NC group (Figure 2(d)). Next, we tested that the iron overload decreased glucose-stimulated insulin secretion, and we found that compared with DIO + sh-NC group, knockdown of XIST could lighten the iron overload-related functional damage on INS-1 cells (Figure 2(e)). e LIP was upregulated in DIO, which was reserved by knockdown of XIST (Figure 2(f )). Furthermore, we detected whether XIST affected the iron metabolic-related proteins, and results indicated that XIST knockdown could downregulate the expression of hepcidin, ferritin, and DMT1 but upregulated TfR1 expression (Figure 2(g)). ese findings showed that XIST may influence the iron metabolism and is involved in iron overload-induced islet beta cells injury.  Computational Intelligence and Neuroscience

XIST Targets and Regulates miR-130a-3p.
Targeting miRNA is one of the regulatory functions of lncRNA. e StarBase predicted that XIST contained complementary binding sites to miR-130a-3p (Figure 3(a)). In previous studies, XIST may act as a competitive endogenous RNA (ceRNA) by inhibiting miR-130a-3p. Hence, we speculated that there is targeting relationship between XIST and miR-130a-3p. To confirm it, a fragment of XIST including the putative WT or MUT binding sites was constructed into the luciferase reporter vectors. As shown in Figure 3(b), the cotransfection with the miR-130a-3p mimics and XIST-WT resulted in an obvious decrease of luciferase activity in HEK 923 cells, while no significant effect was present in cell transfected with the miR-130a-3p mimics and XIST-MUT.

ALK2 Is a Target mRNA of miR-130a-3p.
ALK2 has been demonstrated to be a direct target of miR-130a [26]. As  shown in Figure 5(a), ALK2 mRNA harbored the binding site of miR-130a-3p. e effects of iron overload, XIST, and miR-130a-3p on ALK2 expression are elucidated. en, we detected the protein level of ALK2 in the animal model and found that ALK2 was upregulated in D and DIO groups compared with the control and IO groups, and DIO group was higher compared with D group (Figure 5(b)). Furthermore, ALK2 expression was inhibited and promoted by miR-130a-3p inhibitors and miR-130a-3p mimics, respectively, while cotransfection with miR-130a-3p mimics and Computational Intelligence and Neuroscience pc-XIST had no significant influence on the protein level of ALK2 ( Figure 5(c)). e result of luciferase assay showed that compared with the control group, the luciferase activity was significantly decreased in cell cotransfected with ALK2-WT and miR-130a-3p mimics, while that in cotransfected ALK2-MUT and miR-130-3p mimics did not significantly change ( Figure 5(d)). Taken together, miR-130a-3p directly targeted ALK2.

XIST Promotes Iron Overload-Induced INS-1 Cells Injury by
Regulating miR-130a-3p/ALK2 Axis. To explore the function of XIST/miR-130a-3p/ALK2 axis in iron overload, the miR-130a-3p mimic, pc-XIST, and sh-ALK2 were cotransfected into INS-1 cells. e expression of ALK2 was reduced by interference of ALK2, which was revered with overexpression of XIST, but ALK2 expression was finally repressed by miR-130a-3p mimics (Figure 6(a)). On the contrary, cell proliferation and survival rate were elevated after interference of ALK2 but reduced with XIST upregulation, which was terminally boosted by miR-130a-3p mimics (Figures 6(b) and 6(c)). Next, the LIP and insulin secretion were measured, as shown in Figures 6(d) and 6(e), respectively. e results indicated that downregulation of ALK2 lowered LIP, which was revered with XIST overexpression but LIP was finally decreased by miR-130a-3p upregulation ( Figure 6(d)). On the contrary, insulin secretion was increased after ALK2 knockdown but decreased with XIST upregulation, which was terminally boosted by miR-130a-3p upregulation (Figure 6(e)). ese results suggest that XIST/miR-130a-3p/ALK2 axis regulates islet beta cells injury from iron overload.
It is well established that hepcidin has emerged as the key regulator of uptake and release of iron in the tissues and cells [27]. Bone morphogenetic protein 6 (BMP6) binds to the ALK2 and AKL3 from a heterodimeric complex previously reported to increase hepcidin transcription expression by inducing phosphorylation of SMAD1/5/8 [26]. erefore, we explored whether ALK2 modulated BMP6/AMAD signaling in iron metabolic. e levels of BMP6 and p-SMAD1/5/8 were decreased after knockdown of ALK2 but were increased with overexpression of XIST, which was terminally repressed by miR-130a-3p upregulation (Figure 6(f)). ese results confirmed the ALK2 positive regulation of BMP6/AMAD signaling. Next, iron metabolic-related proteins level was measured by using Western Blot. e expressions of hepcidin and DMT1 were reduced by interference of ALK2, which were revered with overexpression of XIST, but hepcidin and DMT1 expression were finally repressed by miR-130a-3p upregulation ( Figure 6(f)). On the contrary, the expression of TfR1 was elevated after interference of ALK2 but reduced with XIST upregulation, which was terminally boosted by miR-130a-3p overexpression. ese results suggest that XIST/miR-130a-3p/ALK2 axis may protect islet beta cells from iron overload by BMP6/AMAD signaling.

Knockdown of XIST Alleviates Iron Overload-Induced Pancreatic Islets/Beta Cells Injury in T2D Rats.
To determine the role of XIST in iron overload-induced pancreatic islets/ beta cells, we silenced XIST expression in the DIO rats (Figure 7(a)). Notably, XIST depletion significantly increased and decreased the expression of miR-130a-3p and ALK2, respectively (Figures 7(b) and 7(c), respectively). Additionally, XIST knockdown remarkably alleviated iron overload-induced body weight loss (Figure 7(d)). By contrast, XIST knockdown significantly inhibited iron overloadinduced blood glucose, serum iron, TIBC, and serum ferritin levels rise (Figures 7(e)-7(h), respectively). e protein expressions of BMP6, SMAD1/5/8, hepcidin, ferritin, and DMT1 were elevated by iron overload-induced but was reversed with knockdown of XIST (Figure 7(i)). On the contrary, TfR1 protein expression was reduced after iron overload-induced, which was reversed with XIST knockdown. H&E staining revealed the well-defined islets of Langerhans, along with intact exocrine pancreatic tissues in control group, seriously lost in DIO group (Figure 7(j), H&E staining). Sections of control group revealed dense reddish brown cytoplasmic immunoreactivity, which was markedly decreased (Figure 7(j), insulin staining). ere was an improvement in the histology of pancreatic and insulin secretion from XIST knockdown treated groups. ese data indicate that XIST knockdown alleviates iron overload-induced pancreatic islets/beta cells injury in T2D rats.

Discussion
Iron is an essential element, but the iron deposition will result in the reduction of the number of islet beta cells and insulin secretion as described in [28], thus, promoting the occurrence and development of diabetes mellitus [29]. In this study, we have shown that iron overload promoted weight loss and increase of postprandial blood glucose by increasing the expression of iron, TIBC, ferritin, hepcidin, and DMT1 and decreasing the expression of TfR1. rough RT-qPCR analysis, we identified XIST upregulated in T2D. Notably, iron overload did not affect XIST expression but promoted XIST expression in T2D. ese observations suggest that XIST plays a critical role in the iron metabolism of T2D.
LncRNAs have been reported to play widespread roles in gene regulation and other cellular processes [30]. LncRNAs exert their functions via diverse mechanisms, including cotranscriptional regulation, modulation of gene expression, scaffolding of nuclear or cytoplasmic complexes, and pairing with other RNAs [31]. Notably, accumulating evidence shows that lncRNAs are implicated in T2D [32] and iron overload [33]. XIST was highly expressed in T2D patients [18] and was activated by iron overload. Aberrant expression of XIST is implicated in diabetes mellitus and its complications [18,34,35]. Here, we show that knockout of XIST expression impairs the iron overload-induced injury and iron metabolism of INS-1 cells, suggesting that XISTmedicated iron metabolism is critical for iron overloadinduced islet beta cells injury.
Studies have shown that lncRNAs can serve as a miRNA ceRNA regulatory network in many diseases [36]. For example, lncRNA ROR could serve as a ceRNA, which regulates islet beta cell differentiation by sponging miR-145 to target Sox2 [37]. In this study, we found that miR-130a-3p Computational Intelligence and Neuroscience was downregulated and determined as a target of XIST and negatively regulated by XIST, thereby inhibiting iron overload and iron overload-induced injury of INS-1 cells. According to reports, miR-130a is upregulated in mouse liver by iron deficiency and targets the BMP receptor ALK2 to attenuate BMP signaling and hepcidin transcription [26]. Interestingly, miR-130a-3p is upregulated in iron deficiency and downregulated in iron overload, suggesting that iron dose regulates miR-130a-3p expression. However, in this study, upregulation of miR-130a-3p changed iron metabolism and ameliorated iron overload-induced INS-1 cells injury. Recent studies reported that miR-130a-3p is lowly expressed in diabetes mellitus and its complications, and upregulation of miR-130a-3p ameliorates the unhealthy condition of renal tubular epithelial and pancreatic beta cells [38,39].
us, we identified miR-130a-3p as an efficient regulator of iron metabolism in T2D.
Furthermore, the miR-130a-3p and its target gene ALK2 were identified as the downstream molecule of XIST. Our findings revealed that the role of XIST/miR-130a-3p/ALK2 axis regulated iron overload and played a role in T2D pathology. ALK2 (also known as ACVR1) is a BMP type-I receptor subtype that plays an important physiological role in the development of bones, muscles, brain, and other   organs [40]. BMP6 binds to the ALK2 and ALK3 from a heterodimer complex to increase hepcidin transcription expression by inducing phosphorylation of SMAD1/5/8 [26]. Hepcidin has emerged as the key regulator of uptake and release of iron in the tissues and cells, suggesting that ALK2 plays a critical role in iron metabolism. MiR-130a is upregulated in mouse liver by iron deficiency and attenuates BMP signaling and hepcidin transcription by targeting ALK2 [26]. Here, we found that XIST can sponge miR-130a-3p to trigger ALK2 expression, leading to the priming of INS-1 cells iron overload. Knockdown of BMP6 and its receptors ALK2/3/6 with siRNA decreases hepcidin transcription [41]. We also found that knockdown of ALK2 promoted iron transport by decreasing hepcidin transcription via inhibited SMAD1/5/8 activation. Interestingly, BMP6 is the primary upstream gene of ALK2 but BMP6 expression was decreased by knocking ALK2 expression, which also was subjected to the feedback regulation of its downstream signaling in T2D. erefore, the feedback regulation in iron overload remains to be further investigated.

Conclusion
In this paper, we have proposed a novel approach and verified its applicability through various experiments. e results showed that XIST overexpression in iron overload rats was associated with a coincident alteration in iron-related parameters. In vitro, increasing XIST level in response to iron-induced was associated with iron metabolism, proliferation inhibition, apoptosis, and reduction of insulin secretion. In contrast, XIST knockdown alleviated above islet β-cell damage induced by iron overload. XIST can promote islet beta cells iron overload and T2D initiation and development through inhibition of ALK2 expression by sponging miR-130a-3p. Our findings reveal that XIST/miR- 130a-3p/ALK2 axis may play a crucial role in the progression of T2D and that targeting this axis may be an effective strategy for treating patients with T2D.
Data Availability e datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Approval
All protocols of animals and experiments were checked and appropriated by Animal Research Ethical Committee of Kunming University of Science and Technology (Kunming, China).

Disclosure
Weiyuan Li and Qiu Feng are co-first authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Weiyuan Li and Qiu Feng contributed equally to this work. e conception of the paper was completed by Weiyuan Li and Qiu Feng, and the data processing was completed by Chenrong Wang, Zhao Yin, Xiaolu Li, and Lei Li. All authors participated in the review of the paper.